The present invention relates to methods of treating disorders by transplantation of grafts derived from developing, non-syngeneic, renal or hepatic, organs/tissues. More particularly, the present invention relates to methods of treating in humans renal disorders via transplantation of porcine 27- to 28-day gestational stage renal grafts, or of allogeneic human 42- to 56-day gestational stage renal grafts. The present invention further particularly relates to methods of treating in humans disorders amenable to treatment via hepatic transplantation using transplantation of porcine 28-day gestational stage hepatic grafts, or of allogeneic human 7-week gestational stage hepatic grafts.
Transplantation of fully differentiated allogeneic kidneys is a widely practiced, life-saving, medical procedure of choice for treatment of numerous highly debilitating and/or lethal renal disorders of major clinical impact. These include major diseases such as renal complications resulting from diabetes or hypertension, cystic kidney disease, obstructive nephropathy and glomerulonephritis. More than 10,000 kidney transplants are performed each year in the United States on patients with end-stage renal disease, at an annual cost estimated to be in excess of $15 billion.
Transplantation of fully differentiated allogeneic hepatic grafts is a widely practiced, life-saving, medical procedure of choice for treatment of numerous highly debilitating and/or lethal hepatic disorders, or enzyme-deficiency disorders of major clinical impact. Disorders amenable to therapy via hepatic transplantation include such major diseases as cirrhosis, viral hepatitis, and hepatocellular carcinoma. In numerous instances of such disorders, restoration of normal liver function is vital for the survival of affected individuals. The liver is the second most commonly transplanted major organ after the kidney. According to the latest U.S. Centers for Disease Control and Prevention sources, cirrhosis remains the 12th leading cause of death for adults in the United States, with 26,225 deaths reported in 1999 and a death rate of nearly 10 cases per 100,000 persons. This accounts for 1.1 percent of total deaths. Furthermore, this number may grossly underestimate the real impact of end-stage liver disease because it does not include acute liver failure or other etiologies that may lead to the need for liver transplantation. Currently, more than 17,000 people in the United States are waiting for liver transplants. According to the United Network for Organ Sharing (UNOS), about 5,300 liver transplantations were performed in the United States in 2002. Hepatocellular carcinoma is the fifth most common malignant disorder and causes nearly 1 million deaths a year worldwide. Other diseases amenable to treatment via hepatic transplantation include various types of deficiencies in enzymes which can be produced by hepatic tissues, such as clotting factor deficiencies resulting in hemophilia.
Allograft transplantation is a therapeutic modality which is associated with critical disadvantages. Standard therapeutic transplantation of renal or hepatic allografts requires obtainment of donor derived grafts which are immunologically, as well as morphologically, matched with the graft recipient. However, the criteria for such matching, particularly the immunological matching, are highly stringent and difficult to fulfill. As such, allografts which are suitably matched to prospective recipients are, in numerous cases, simply unavailable. Thus, large numbers of patients who would otherwise benefit from therapeutic allograft transplantation succumb to diseases associated with organ failure, while awaiting matched transplant donors. In the case of kidney transplantation, approximately eight to nine patients die every day while waiting for a transplant due to the shortage of donors. While each year in the United States, there are an estimated 25,000 potential donors who die, of this number, only about 5,000 have made arrangements to donate their organs. In 1996, of the 10,017 kidneys recovered for transplant, 12 percent failed to meet the donor criteria for transplantation. For example, the average waiting period for obtaining a suitable cadaveric kidney may be more than two years, and only 15 to 20 percent of patients waiting for a transplant receive them. However, even following optimally successful allograft transplantation, permanent and daily administration of toxic doses of immunosuppressive drugs such as cyclosporin A is mandatory to prevent graft rejection. Administration of drugs such as cyclosporin A is highly undesirable since such drugs are associated with severe side-effects, including carcinogenicity, nephrotoxicity, and lead to increased susceptibility to opportunistic infections. Such immunosuppressive regimens are furthermore often unsuccessful in preventing allograft rejection in the medium term, and in any case generally cannot indefinitely prevent graft rejection in the long term. Current allograft transplantation methods are generally performed by harvesting allografts from living human donors, thus requiring subjecting healthy human donors to organ loss via potentially fatal major surgery. While cadaveric graft donors are widely employed, their use presents ethical dilemmas for donor family members as well as for recipients, and is associated with lower success rates than use of living donors. As a last resort back-up alternative to renal transplantation, permanent hemodialysis can be used to sustain life in the case of kidney failure, however this procedure is highly debilitating, cumbersome, expensive, of limited effectiveness, and is associated with a significant risk of opportunistic infections.
The use of xenografts, in particular porcine xenografts has been proposed as a means to overcome the shortage of available human organs for transplantation. Porcine grafts are widely considered to be the ideal animal alternative to human grafts for therapeutic transplantation in humans due to their morphological compatibility with the human anatomy, and due to their essentially unlimited supply which would overcome the restricted availability impediment inherent to prior art human grafts (Auchincloss, H. and Sachs, D. H., 1998. Annu. Rev. Immunol. 16, 433-470). The use of such animal grafts would present the advantage of circumventing the medical/ethical burdens of harvesting grafts from human donors. However, to date no methods of xenograft transplantation have been devised which are capable of overcoming the rapid and vigorous immune rejection of xenografts by the host immune system following transplantation.
Thus, novel and optimal methods of therapeutic renal or hepatic allograft/xenograft transplantation which overcome the limitations of the prior art are urgently required.
It has been known for over four decades that grafts derived from developing organs/tissues are less immunogenic following transplantation into non-syngeneic hosts than grafts derived from corresponding fully differentiated organs/tissues (Medawar, P. B., 1953. Symp. Soc. Exp. Biol. 7, 320-323). Subsequent studies, such as those using a human to rat xenogeneic renal transplantation model (Dekel B. et al., 1997. Transplantation 64, 1550; Dekel B. et al., 2000. Transplantation 69, 1470), or an allogeneic rat renal transplantation model (Hammerman M R., 2000. Pediatr Nephrol. 14, 513) have confirmed these observations. Various mechanisms have been suggested to explain the reduced immunogenicity of developing tissue grafts. It has been suggested that such developing tissue-derived grafts induce attenuated host anti-graft immune responses compared to adult stage tissue-derived grafts due to the former being predominantly vascularized by host-derived vasculature, as opposed to the predominantly graft-derived graft vascularization observed in the latter (Hyink D. P. et al., 1996. Am J. Physiol. 270, F886). It has further been suggested that the low levels of major histocompatibility (MHC) and adhesion molecule expression, and of antigen presenting cells in gestational stage tissue grafts decreases the capacity of such grafts to activate host immune responses.
Thus, a potentially optimal strategy for performing therapeutic organ transplantation in humans would be to use grafts derived from developing allogeneic human, or from developing xenogeneic porcine organs/tissues As well as having potentially optimally low immunogenicity, such grafts would potentially provide the further advantage of inherently possessing optimal growth and differentiation potentials relative to those derived from fully differentiated organs/tissues. As such, such developing organ/tissue grafts may have optimal capacity, following transplantation into a recipient, for generating graft-derived organs/tissues which are morphologically and functionally integrated with the recipient.
In the developing human kidney, fresh stem cells are induced into the nephrogenic pathway to form nephrons until 34 weeks (238 days) of gestation. Such nephrogenic differentiation pathway involves invasion of a specialized region of intermediate mesoderm by an epithelial source (ureteric bud), which grows and branches to form a collecting duct system, and induces disorganized metarenal mesenchymal stem cells to group and differentiate into nephrons [Woolf, A. S. in: Pediatric Nephrology, 4th ed. Barratt, T. M., Avner, A. and Harmon, W. (eds.), Williams & Wilkins, Baltimore, Md. pp. 1-19 (1999)].
Various approaches for using grafts derived from non-fully differentiated sources have been suggested or attempted in the prior art.
One general embryonic stem (ES) cell approach involves culturing human ES cells, which are pluripotent, so as to produce cell types/tissues of a desired embryonic germ layer, and to employ such cells/tissues as therapeutic grafts (Thomson J A. et al., 1998. Science. 282:1145-7; Reubinoff B E. et al., 2000. Nat Biotechnol. 18:399-404). This approach, however, has failed to provide renal or hepatic grafts suitable for therapeutic transplantation. Furthermore, it was found that transplantation of cultured ES cell grafts into immunocompromised mouse hosts generate teratomas (Reubinoff B E. et al., 2000. Nat Biotechnol. 18:399-404), a highly undesirable potentially harmful consequence in the therapeutic transplantation context.
One ES cell/hepatic approach involves genetically modifying mouse ES cell lines to express hepatocyte nuclear factor (HNF)-3beta (Ishizaka S. et al., 2002. FASEB J 16, 1444-1446) so as to generate cultured ES cell-derived hepatocytes for transplantation.
Another ES cell/hepatic approach involves selecting for transplantation hepatocytes from cultured ES cells genetically modified with a gene trap vector insertion into an ankyrin repeat-containing gene providing a beta-galactosidase marker of early differentiation of hepatocytes in-vitro (Jones E A. et al., 2002. Exp Cell Res. 272, 15-22).
The prior art ES cell/hepatic approaches, however, not attempted transplantation of such cultured ES cell-derived hepatocyte grafts in a host, and have therefore failed to demonstrate that following transplantation into a non-syngeneic host, such grafts will be well tolerated by the host, will not generate teratomas/undesired lineages, and/or will provide hepatic functionality.
Various prior art approaches have been proposed for using grafts derived from developing kidneys for performing non-syngeneic renal transplantation.
One renal/allogeneic approach involves transplantation of rat 15-day gestational stage renal grafts under the renal capsule or into the omentum of rat hosts (Rogers, S. A. et al., 1998. Kidney Int. 54, 27-37; Rogers, S. A. et al., 2001. Am. J. Physiol. Regul. Integr. Comp. Physiol. 280, R132-136; Rogers, S. A. and Hammerman, M. R., 2001. Am. J. Physiol. Regul. Integr. Comp. Physiol. 281, R661-665; U.S. Pat. No. 5,976,524 to Hammerman).
Another renal/allogeneic approach involves transplantation of rat 15- or 17-day gestational stage renal grafts into the anterior eye chamber or under the kidney capsule of allogeneic adult rat hosts. While such transplanted grafts became vascularized and displayed renal differentiation after 9-10 posttransplantation, by 16 days posttransplantation they exhibited obvious signs of graft rejection, including generation of hypercellular glomeruli and lymphocytic infiltration in peritubular spaces (Abrahamson et al., 1991. Lab. Invest 64:629-639).
A further renal/allogeneic approach involves transplantation of mouse 12-day gestational stage renal grafts previously subjected to multi-day organ culture into the anterior eye chamber or renal cortex of allogeneic newborn or adult recipients (Robert et al., 1996. Am. J. Physiol. 271:F744-F753). In this approach, by 7 days post-transplantation, grafts implanted in both newborn and adult hosts had a vascular component which was significantly of host origin, a factor which strongly correlates with eventual graft rejection.
An additional renal/allogeneic approach involves transplantation of sections of rat 1-day old neonatal or 15- to 17-day gestational stage renal grafts into related or unrelated allogeneic recipients. However, following transplantation, lymphocytic infiltration of grafts and replacement of the grafts by fibrosis occurred in both related and unrelated adult hosts, and was more rapid in the unrelated hosts (Barakat and Harrison, 1971. J. Anat. 110:393-407).
One renal/xenogeneic approach involves transplantation of rat 15-day gestational stage renal grafts into the omentum of mouse hosts subjected to CTLA4-Ig costimulation blockade for prevention of graft rejection (Rogers, S. A. and Hammerman, M. R., 2001. Am. J. Physiol. Regul. Integr. Comp. Physiol. 280, R1865-1869).
Another renal/xenogeneic approach involves transplantation of human 98- to 154-day gestational stage renal grafts into chimeric rats bearing human PBMCs (Dekel B. et al., 1997. Transplantation 64, 1550), or transplantation of human 70-day gestational stage renal grafts into immune deficient mice (Dekel B. et al., 2000. Transplantation 69, 1470).
A further renal/xenogeneic approach suggests transplantation of “approximately” 20- to 30-day gestational stage porcine renal grafts (U.S. Pat. No. 5,976,524 to Hammerman). This approach, however, is highly speculative by virtue of never having been experimentally tested, and therefore fails to demonstrate that, following transplantation into a non-syngeneic host, grafts at such gestational stages will be well tolerated by the host, will generate developed, functional renal organs/tissues, and will not generate teratomas/undesired non-renal lineages.
Various prior art approaches have been proposed for using grafts derived from developing liver for performing non-syngeneic hepatic transplantation.
One hepatic/xenogeneic approach involves transplantation of embryonic porcine hepatocytes into the spleen of immune deficient rats (Kokudo N. et al., 1996. Cell Transplantation 5:S21-2). In these experiments, hepatic function in graft recipients was analyzed at four weeks posttransplantation.
Another hepatic/xenogeneic approach involves transplantation of porcine fetal liver fragments enclosed in microporous immunoisolation capsules into the omentum of rat recipients having acute hepatic failure (Takebe K. et al., 1996. Cell Transplant 5:S31-3). Such transplantation, however, was found to be associated with an unacceptably high death rate in graft recipients.
A further hepatic/xenogeneic approach involves transplantation of fetal or neonatal porcine liver fragments enclosed in microporous immunoisolation capsules into dog recipients having hepatic failure (Kanai N. et al., 1999. Cell Transplantation 8:413-7). In these studies the grafts were examined histologically 14 days posttransplantation.
An additional hepatic/xenogeneic approach involves transplantation of porcine very late-stage fetal liver tissue into dogs (Kanai N. et al., 1999. Transplant Immunology 7:95-9). In these experiments, hyperacute graft rejection was only delayed, as compared to that occurring following transplantation of adult-stage grafts, but not prevented.
One hepatic/allogeneic approach involves transplantation of rat fetal liver into the spleen of rats subjected to FK506 immunosuppression (Kokudo N. et al., 1996. Cell Transplantation 5:S21-2). These studies suggested that immunosuppressive recipient treatment was required for achieving engraftment until four weeks posttransplantation.
All such prior art approaches involving use of grafts derived from developing kidney or liver have significant disadvantages, including: undemonstrated or suboptimal short- and/or long-term immune tolerance by graft hosts, and/or requirement for graft host immunosuppressive treatment; undemonstrated or suboptimal short- or long-term structural and functional graft differentiation into functional renal or hepatic organs; predominantly graft-derived, as opposed to host-derived, graft vascularization following transplantation, the former strongly correlating with risk of eventual graft rejection; inadequate availability of transplantable grafts; failure to demonstrate that the grafts employed are at a sufficiently advanced developmental stage to avoid teratoma/undesired tissue lineage differentiation following transplantation, and hence failure to demonstrate safety for therapeutic applications. Xenogeneic approaches involving non-porcine organ grafts fail to provide grafts which can be transplanted in humans. Approaches involving encapsulated hepatic grafts fail to provide hepatic grafts capable of providing any of the numerous critical hepatic functions requiring free contact between the liver and circulating cells/particles.
Thus, all prior art approaches have failed to provide an adequate solution for using transplantation of developing non-syngeneic renal or hepatic grafts to treat human disorders amenable to treatment via transplantation of such grafts.
There is thus a widely recognized need for, and it would be highly advantageous to have, a method of treating human disorders via transplantation of non-syngeneic developing renal or hepatic grafts devoid of the above limitation.